Presented at:
American Transplant Congress, Boston, MA, May 16, 2004

Immunologic Assays in Patients Successfully Withdrawn from Immunosuppression (IS) after Liver Transplantation

G. V. Mazariegos, A. Zahorchak, J. Reyes, H. Chapman, A. Girnita, K. McDade, A. Zeevi, A. Thompson

Thomas Starzl Transplantation Institute, Pittsburgh, PA


Aim: The long term results of patients successfully withdrawn from IS and preliminary "tolerance" assay testing in this cohort are presented. We hypothesized that tolerant patients would be more likely to exhibit plasmacytoid DC lineage (pDC2), lack donor specific alloantibody, and demonstrate genotypes associated with low producers of pro-inflammatory cytokines.

Methods: Patients completely withdrawn from IS (Group A) between June 1992 and June 2002 were considered for tolerance assay testing consisting of dendritic cell subsets, donor specific alloantibody, and cytokine gene polymorphism. Patients undergoing prospective drug withdrawal (Group B) and those who required maintenance immunosuppression because of failed weaning or rejection history (Group C) were also studied. Assay methods were as follows:

• DC Assay Dendritic cells (DC) were identified as HLA-DR positive (+) and lineage (lin) marker (CD3, CD14, CD19, CD20) negative (-) by four-color flow cytometric analysis. Precursor (p) DC2 and (p) DC1 subpopulations defined respectively as HLA-DR + , lin - , CD123 hi (IL3R a hi ), CD11c - and as HLA-DR + , lin - , CD123 lo (IL3R a lo ), CD11c + were further quantified.
• Alloantibody by ELISA was performed for the presence (ELISA+) and donor-specificity of HLA antibodies (DS HLA Ab).
• Cytokine Polymorphism Allelic variations in tumor necrosis factor (TNF)-a and IL-10 were determined using PCR-SSP technique with commercially available kits. Statistical analysis was performed by X 2 tests with 2 X 2 analysis for alloantibody and polymorphism data with 2-tailed Mann Whitney U test for DC data analysis. P<0.05 was considered significant.

Results: 25 patients completely withdrawn from medication by physician directed protocol (n=13), emergently for life-threatening indications (n=8), or because of patient non-compliance (n=4) have maintained normal liver function and freedom from IS for a mean of 7.6+/- 5.7 years (range 1.2-21.7).

82 patients (25 Group A, 35 Group B, and 22 Group C) underwent testing as described. DC and alloantibody results are depicted below.

#p<0.05 between Groups A and B compared to C

*P<0.05 Group A versus Group C (X2 = 5.86, p < 0.05)

Although the number of patients with both TNF-a low and IL-10 high/intermediate polymorphism was slightly greater (19/25, 76%) in the tolerant group (A) than the maintenance group (C) (13/20, 65%), this difference was not statistically significant.

Conclusions Clinically tolerant liver transplant recipients demonstrated higher pDC2/pDC1 ratio, and lacked donor specific antibody as compared to patients who required ongoing IS. The DC subset ratio was similar to those undergoing prospective weaning (Group B) but the groups differed in incidence of DS HLA Ab. Results of prospective weaning may help determine the relative importance of each of these findings and contribute to immunologically characterizing the tolerant patient.

Acknowledgment: This research was performed as a project of the Immune Tolerance Network, a seven year clinical research project headquartered at the University of California San Francisco and supported by the National Institute of Allergy and Infectious Diseases, the National Institute of Diabetes, and Digestive and Kidney Disease and the Juvenile Diabetes Research Foundation

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